A unique cell-free system from Saccharomyces cerevisiae, that translates endogenous messenger RNAs as well as exogenous natural mRNAs and synthetic polynucleotide templates, has been developed in this laboratory; translation is carried out actively and accurately. A strategy and the methods of procedure that allow for the analysis of most of the individual reactions involved in polypeptide chain initiation, elongation, and termination, have also been developed. A number of temperature-sensitive mutants of yeast, with altered components in the protein biosynthetic pathway are available in this department. Four such mutants are currently under investigation. Preliminary studies indicate that in two of these mutants, a component required for chain initiation is altered; although both mutations involve initiation components, they are different. In two other mutants, a component involved in chain elongation appears to be altered. Using the procedures available in this laboratory, the altered components (translational factors or ribonucleoprotein particles) will be localized in the pathway and identified. If the temperature-sensitive components are protein factors, they will be purified and characterized chemically, structurally, and biologically as to the specifically altered function; isolation of sufficient pure factor for sequencing would be carried out. If the temperature-sensitive components are ribonucleoprotein particles, such as 80S ribosomes, 60S subunits or 40S subunits, the altered ribosomal protein or nucleic acid will be identified, purified and characterized. The effects of such genetic blocks, on other steps in translation or on transcription and other mutants, affecting macromolecule metabolism, will also be examind.